Searching for microbes Part II. Culture of bacteria & yeasts

1. Searching for microbesPart II.Culture of bacteria & yeasts Ondřej Zahradníček to practical of VLLM0421c contact to me: zahradnicek@fnusa.cz

2. Survey of parts of this slideshow Multiplication of bacteria, growth curve Conditions needed for bacterial (or fungal) culture Bacterial culture – introduction Liquid media Solid media: inoculation of a specimen / strain Solid media: classification and examples Questions for ROPOT

3. Tale • It was once a bacterium, and it was very small, and so without the microscope nobody has seen it, and in the microscope it was very difficult to see its shape. It was very unhappy, because it emphasized very much its beauty. • Once, Mr. Koch came. He put the bacterium onto asolid medium and hide it into a thermostat. Bacterium was very happy and quickly started to multiply…

4. And Day After… • Mr. Koch came, opened the thermostat, took out the Petri dish with medium, and he saw: his liked, tiny bacterium was visible by a naked eye! Of course, not as one bacterium, but as a strain of totally identical cells, that raised from that bacterium in form of a colony • The bacterium was very happy, and it showed its beauty to mr. Koch. It showed him its shape, size, profile of its colony, pigments and many other things.

5. Multiplication of bacteria, growth curve

6. Multiplication of bacteria • Each bacteria has its generation period • During one gereration period one makes two, in ten times of the period one makes 1024 bacteria (theoretically) and so on,. • Ideal multiplication would exist only if we would add all the time nutrients and eventually oxygen and we would remove waste products. • At usuall conditions (when we do not no that) the real growth curve should be used – see the next slide

7. Real growth curve • Phase of latence – we let bacteria grow, but they still do not multiply • Exponencial phase – growth accelerates • Stational phase– they grow with the same speed all the time • Slowing and stopping of growth – lack of nutrients, to many waste, or bacteria regulate themselves by „quorum sensing“

8. Conditions needed for bacterial (fun- gal) culture

9. Do the conditions for bacterial growth matter? Of course they do! Majority of bacteria need their temperature, moisture, salts concetration and many other characteristics to be in a quite narrow range. lower survival limit (bactericidal) lower growth limit (inhibitory) lower growth limit (inhibitory) upper survival limit (bactericidal) Values, that enable microbial survival, are not sufficient. They should be able to multiply. Various microbes need various conditions!

10. Medically important bacteria • Temperature usually needed around 37 °C • but bird pathogens more (42 °C), microbes coming from outer environment less (30 °C) • Value of pH needed around pH 7 • but gastric helicobacter by far less • Water amount should be high, especially for G– • Osmolarity usually described as NaCl concentration, that is usually around 0,9 % (physiological saline) • but staphylococci, that have to be able to multiply on sweated skin, multiplies even at 10 % of salt! In practice part of parameters (e. g. temperature) is derived from thermostat settings, and remainder (e. g. NaCl concentrations) by composition of the culture medium.

11. Culture thermostat (incubator) Besides box thermostats, like this one, our Institute has a chamber thermostat, too. It is a whole room with 37 °C. Majority of bacteria is cultured in a thermostat overnight, so about 24 h. Photo O. Z.

12. Example of use of relation to temparature in bacterial diagnostics • Pseudomonas aeruginosa grows at 37 °C and 42 °C. • On the other hand, Pseudomonas fluorescens grows at 4 °C and 37 °C. Besides Pseudomonas, Listeria, too, and yeasts and molds grow at lower temperature. Elevated temperature is suitable e. g. for Campylopacter

13. 10 % 6,5 % Influence of NaCl concentrations to the growth of some bacterial genera Majority of Enterococci Staphylococci bacteria

14. Besides various NaCl concentrations • Adding of sodium azide enables growth of enterococci, but neither staphylococci, nor streptococci are able to grow • Amikacin enables growth of streptococci and enterococci. Staphylococci, sensitive to amikacin, are eliminated.

15. A little about catabolism of bacteria • We have three types of catabolism: • Fermentation – nutrients breakdown without need for oxygen. Little energetically effective, but does not need oxygen. Forduct is e. g. lactate, ethanol etc. • Aerobic respiration – little nutrients gives a lot of energy, but oxygen is needed. Product is CO2 and H2O • Anaerobic respiration – another electron acceptor • Type of catabolism is closely connected with relation to oxygen.Fermentating bacteria are usually facultatively or strictly anaerobic. On the other hand, aerobically respirating bacteria use to be strictly aerobic.

16. Relation to oxygen often together described as just simply „aerobes“ • Strict aerobes grow only in the presence of oxygen • Facultative anaerobes and aerotolerant bacteria (it is not possible to differentiate them) grow at all athmosphere conditions • Strict anaerobes they grow only in environment without oxygen • Microaerophile bacteria grow only in conditions with traces of oxygen • Capnophile bacteria need more CO2

17. Growing anaerobic bacteria www.medmicro.info www.medmicro.info

18. Bacterial culture – introduction

19. Why we culture bacteria • Why bacteria are cultured in the laboratory? • To keep them living and to multiply them. This is gained by cultivation in both liquid and solid media (jelly-consistence media, based on agar algae) • To obtain strain – solid media only; invented by Robert Koch • To differentiate and divide them mutually –diagnostic and selective media are used, for identification

20. Specimen and strain • Specimen is taken from a patient. Specimen contains cells macroorganism, various number of microbial species (zero to maybe twenty) and more items • A strain – an isolate – is a population of one bacteria, isolated from a specimen on a solid medium • To gain a strain, we have to grow a bacterium on a solid medium and inoculate carefully

21. Term „colony“ • A colony is a formation on a surface of a solid media. It is developped from one cell or a small group (couple, chain, cluster) • In some cases number of colonies on an agar shows us number of microbes in the specimen – or more preciselly, number of „colony forming units“ (CFU) • Description of colonies has an important place in.bacterial diagnostics www.medmicro.info

22. Solid media www.medmicro.info

23. Is it good, or bad, that various bacteria need different conditions? • It is bad, because it complicates deffinition of conditions, that would be suitable for majority of bacteria • It is good, because this enables to use it in diagnostics (e. g. growth ability on medium with 10 % NaCl differenciates well stafylococci)

24. Media globally versus media in medical microbiology • In industrial microbiology or in some other applications we use mostly chemically defined media. We know their composition, and it is possible to observe, how much of something increased or decreased. • In clinical microbiology we have no need to know a detailed composition. Often some parts of media are not definable (red blood cells, yeast extract).

25. Liquid media and solid media • Liquid media are based on je meat-peptonic broth (exctract of cooked beef meat + protein hydrolysate). They are used mostly to multiplication. It is difficult to evaluate the result, in fact, only „non turbid broth – turbid broth“ (growth – no growth) • Majority of solid media are based on the same broth, but supplied by an agar alge extract. Bacteria grow slower on solid media, but the result is very variable, and it is possible to get a strain.

26. Various specimens – various cultivation • How the specimen type influences culture? • Specimens, where microbes use to be rare are inoculated into liquid media only. Microbes multiply quickly. Example: conjunctival swab • Specimens, where the amout of microbes may vary, but even small amounts are important are cultured on both liquid and solid media. Example: wound swab • Specimens, where usually we have many microbes, eventually even common microflora, are cultured on solid media only. Example: throat swab

27. Liquid media

28. Liquid media www.medmicro.info

29. Classification of liquid media • Liquid media have two categories only: • multiplyingmediaare common and universal. Example: broth for aerobic culture and VL-broth for anaerobic culture (VL = viande-levure, from french – contains meat-yeas extract) • selectively multiplyingmediawere developped to multilply some bacteria and to supress multiplication of other. Example: selenite broth for salmonella

30. Solid media: inoculation of specimen and strain

31. Solid media www.medmicro.info

32. Solid (agar) media • To have all advantages, given by solid media, we have both the specimen musíme specimen (cultivation specimen  strain), but also strain (cultivation strain  strain) dilute properly at inoculation. Classical way of dilution inoculation is so named cross inoculation. In practice, usually e. g. one halfth of a plate is inoculated by a swab, and then diluted by a loop. Sometimes some discs and culture lines are added – not being a topic for today.

33. Why an isolated colony is so important • Only ao we can identify larger number of mixed pathogens • But also because only isolated colonies enable to observe typical colony characteristics. The best clown is not able to show you his art, when kept with many other clowns in a small cupboard.

34. In case of a mixture, each bacterium forms its own colonies(at a proper dilution inoculation) 1 – inoculation of bacterial mixture (dots), 2 – result of cultivation: in first parts of inoculation a mixture, at the end – isolated colonies

35. How to inoculate of a specimen to a medium Using the swab, inoculate the on a part of the agar plate (to about one third of Petri dish diameter) Sterilize your loop Dilute from part with specimen, making the second part of inoculation Sterilize your loop Dilute from lines inoculated in the second phase (not touching the part inoculated by the swab) Sterilize your loop Inoculate the „serpent“ on the remaining part of the plate

36. How to reinoculate of an agar culture Sterilize your loop Take the strain Inoculate first phase Sterilize your loop Do not take the strain again Inoculate second phase Sterilize your loop Do not take the strain again Inoculate third phase Sterilize your loop Do not take the strain again Inoculate the „serpent“

37. Size Colour Shape (round…) Forfile (convex…) Edges Surface (smooth, rough…) Consistence (dry…) Transparency Smell Colony surroundings* What to describe at colonies *Definition is related to the medium used. For example, haemolysis is observed around some bacteria grown on media with RBCs.

38. Difference between shape and profile

39. Solid media: classification and examples

40. Solid selective media • They have to select (separate) from a bacterial mixtureonly one of several groups of genera • An example is blood agar with 10 % NaCl used for stafylococci • Sometimes, selectivity is reached by an antibiotic addition. Blood agar with amikacin is selective for streptococci and enterococci

41. Photo O. Z. Diagnostic media • They do not supress growth of any microbe • On the other hand, their composition enable them to differenciate microbes according to some properties • An example is blood agar to observe haemolytical properties, and VL blood agar (simillar, but to anaerobes). WCHA agar is a variant of VL blood agar. • Special case are chromogenic and fluorogenicmedia Photo O. Z.

42. Changes on blood agar • All media with RBCs (blood agar, VL bloodní agar, agar with washed RBCc etc. –but not blood agar with 10 % NaCl, where RBCs are lyzed) enable us to see: www.medmicro.info • Total haemolysis • Partial haemolysis • Absence of haemolysis • Viridation (green)

43. Chromogenic and fluorogenic media • Chromogenic media contain a dye with bound specific substrate  it loses it colour, it is no more a dye, but a chromogen • bacteria able to breakdown the specific substrate change the chromogen againt to the original dye • The medium may contain more chromogens (for more species) • Fluorogenic media: similar, with a fluorescent dye www.oxoid.com

44. Chromogenic media for yeasts Four various yeasts that grow in typical colonies – one in.green, one in.blue, one in.dry pink and one in smooth pink. Other yeasts are white on this medium www.medmicro.info

45. www.medmicro.info Properties of Endo agar • Endo agar enable growth of G- bacteria only, and and even not all of them (selectivity) • The growing bacteria can be differenciated into lactose positive (red) and negative (pale). • From the point of view of medical microbiology, it is important: lactose positive bacteria are usually milder pathogens than lactose negative bacteria • A simillar is McConkey medium, more common in world (but not used in OUR laboratory) • Selective diagnostic are also XLD, CIN media etc.

46. XLD and MAL media for Salmonella www.medmicro.info

47. From our chamber refrigerator www.medmicro.info

48. Selective, diagnostic and selective diagnostic media – review

49. Enriched and selective enriched media • For bacteria with specific need for nutrients • They are enriched by different chemicals • Even blood agar is an enriched medium, although shown as a diagnostic medium (it may be considered a member of both groups). • An expample of „pure enriched medium“ is chocolate agar for pathogenous species of Neisseria and Hemophilus (these do not grow even on blood agar). Filtrated chocolate agar is known as Levinthal agar or simply „Haemophilus agar“ (HEM); it is only suitalbe for Haemophilus, not Neisseria • Media may be selective enriched – e. g. GC agar, – chocolate agar for culture of Neisseria gonorrhoeae that also contains antibiotics to suppress other macteria

50. Chocolate agar www.medmicro.info